Long, noncoding RNAs (lncRNAs) have diverse roles in a variety of cellular processes, including stem cell pluripotency, cell cycle control, and immune regulation. LncRNAs function by varying mechanisms, and have the capacity to coordinately regulate the activity of many genes at once. Surveys of the human genome indicate the presence of thousands of lncRNAs. The vast majority have not yet been studied in detail. The goal of this Ruth L. Kirschstein NRSA Postdoctoral Foundation (F32) application is to characterize the role of lncRNAs in epithelial differentiation. Initial work in our laboratory has identified a new LncRNA that is upregulated during keratinocytes differentiation, which has been named terminal differentiation-induced noncoding RNA (TINCR). Depletion of this LncRNA in an epithelial model results in a differentiation defect. RNA-protein interaction assays demonstrate that TINCR interacts with Staufen-1 (Stau1) which is an RNA-binding protein with known roles in RNA trafficking and degradation. Depletion of Stau1 results in an epidermal differentiation defect that phenocopies TINCR depletion. These findings support the potential role of a new LncRNA and its interacting protein in skin differentiation, and provide a framework to further explore their functions in the skin. The first objective of this proposal is to define the functionl domains of TINCR. Using a series of deletion mutants, the function of individual segments of this new LncRNA will be assayed in an organotypic skin model. The second objective of this proposal will be to determine the relationship between TINCR and Stau1. Epistasis experiments will be performed to determine how these elements interact genetically to regulate skin differentiation. Then, specific molecular models of their function will be tested using RNA-interference based genetic knockdown experiments. A role for lncRNAs in the homeostasis of somatic tissues, such as skin, has not yet been described in the literature. The lines of experimentation described in this proposal will provide insight to the mechanism of action of a novel LncRNA, TINCR, in the regulation of skin homeostasis and differentiation. Dysregulated skin differentiation is a hallmark of several skin diseases, and improving the knowledge of the molecular regulators of the differentiation process will facilitate approaches to understand the cause and potential treatment of these conditions.